Reverse transcriptase inhibitors for treating adenocarcinomas

ABSTRACT

A reverse transcriptase inhibitor such as 3 min - azido - 3 min  - deoxythymidine is used for the treatment or prophylaxis of a retrovivus-associated carcinoma such as breast cancer in a human.

The present invention is concerned with materials and processes for thetreatment of human adenocarcinomas, particularly breast carcinoma.

Breast carcinoma is known to affect about 9% of women in the WesternWorld, and in women aged between 40 to 54 years it is the major cause ofdeath.

Monocytes from patients with breast cancer show depression of bothdirectional and random migration, and lower phagocytic activity comparedwith those obtained from control subjects. This dysfunction isassociated with giant-cell formation when such cells are incubated for 6days. Virus-induced cell fusion is a possible means of giant-cellformation. Giant-cell formation can be induced in normal monocytes byincubation with a 220 nm filtrate of cytosol or a similar filtrate ofcell-free culture medium (CFCM) obtained from the incubated monocytesfrom patients with breast cancer. These observations strongly suggestthat monocytes from patients with breast cancer contain a factor thathas the ability to induce giant-cell formation by monocytes from normalcontrols.

In mice, the development of one particular form of mammary tumourdepends on the presence of a retrovirus, murine mammary tumour virus(MMTV). Some homology has been reported between proviral DNA sequencesof MMTV and the human genome, and one suggestion has been that thehomology is due to the presence of an as yet unidentified latentretrovirus in certain human tissues. The human breast cancer cell line(T471) has been reported to contain a gene sequence (9 kb long) that hasa homology with part of the genome of MMTV. These findings havereawakened old speculation that human breast cancer may have, at leastin part, a viral aetiology.

Recently some very specific anti-viral materials have become available.These are zidovudine (also referred to as "AZT" and chemically3'-azido-3'-dexoythymidine), dideoxyadenosine ("DDA"), dideoxycytidine("DDC") and phosphoformate (`Foscarnet`). They all have the specificproperty of inhibiting the reverse transcriptase enzyme of retroviruses,and thus could be used to determine whether such enzyme is present andhence to establish specifically the presence of retroviral entities.

It has now been found that monocyte cells in human breast cancer doindeed contain at least one retroviral reverse transcriptase.Furthermore it has now been found that retroviral activity is present incells other than monocytes and that it can be transmitted from cell tocell. It has moreover also been found that the activity of such, andtherefore of the retroviruses themselves, can be inhibited by the use ofzidovudine and close derivatives and congeners thereof.

According to the present invention we provide the use of a reversetranscriptase inhibitor or a pharmaceutically acceptable derivativethereof in the manufacture of a medicament for the treatment orprophylaxis of retrovirus-associated adenocarcinoma in a human; and/orthe treatment or prophylaxis of a human retrovirus infection associatedwith adenocarcinoma in a human.

According to further features of the present invention we provide:

a) a method for the treatment or prophylaxis of retrovirus-associatedadenocarcinoma in a human which comprises the administration of reversetranscriptase inhibitor or a pharmaceutically acceptable derivativethereof to the human in an amount effective to treat saidadenocarcinoma;

b) a method for the treatment or prophylaxis of a retrovirus infectionassociated with adenocarcinoma in a human which comprises administeringto the human a reverse transcriptase inhibitor or a pharmaceuticallyacceptable derivative thereof in an amount effective to treat or inhibitsaid retrovirus infection; and

c) a method of alleviating the symptoms of a retrovirus-associated humanadenocarcinoma which comprises the administration of a reversetranscriptase inhibitor or a pharmaceutically acceptable derivativethereof to the human in amount effective to alleviate said symptoms.

The present invention also includes a method for the identification ofretrovirus-associated human adenocarcinoma which comprises bringing abiological sample of, or derived from, human tissue into contact with anagent adapted for the identification of retrovirus.

A method for the identification of retrovirus-associated humanadenocarcinoma which comprises bringing a biological sample of, orderived from, human tissue into contact with an agent adapted for theidentification of retroviral reverse transcriptase.

With regard to the above-described treatment of a retrovirus associatedwith adenocarcinoma, the present invention includes specifically theinhibition of the reverse transcriptase of the said retrovirus.

It will be understood that any known reverse transcriptase inhibitor maybe used in accordance with the present invention. Furthermore it will beunderstood that the present invention also provides for the use of morethan one reverse transcriptase inhibitor either simultaneously or inconjunction. Examples of reverse transcriptase inhibitors include3'-azido-3'-deoxythymidine and other 3'-azido purine or pyrimidinenucleosides, for example as described in EP 217580 (Wellcome)2',3'-dideoxy purine nucleosides such as 2',3'-dideoxy-2-amino-purine,3'-fluoronucleosides such as 3'fluoroguanosine, carbocyclic nucleosidessuch as carbovir, 2',3'-dideoxy-2',3'-didehydro nucleosides such as2',3'dideoxy-2',3'-didehydrothymidine, and ribavirin.

A preferred inhibitor of reverse transcriptase is3'-azido-3'-deoxythymidine.

The present invention is particularly useful in relation to human breastcarcinoma.

According to a specific embodiment of the present invention we provide.

The use of 3'-azido-3'-deoxythymidine or a pharmaceutically acceptablederivative thereof in the manufacture of a medicament for the treatmentor prophylaxis of breast carcinoma in a human; and/or the treatment orprophylaxis of a human retrovirus infection associated with breastcarcinoma in a human.

The term "pharmaceutically acceptable derivative" is used herein todenote a physiologically functional equivalent of3'-azido-3'-deoxythymidine and includes any pharmaceutically acceptablesalt or ester (or salt of such ester) of 3'-azido-3'deoxythymidine orany other compound which, upon administration to a human subject, iscapable of providing (directly or indirectly) 3'-azido-3'-deoxythymidineor an antiretrovirally active metabolite or residue thereof. Thus, forexample, it is understood that 3'-azido-3'-deoxythymidine isphosphorylated in vivo successively to the monophosphate, thediphosphate and finally to the triphosphate ester which acts as aninhibitor of the reverse transcriptase of the retrovirus which has beenidentified in association with breast carcinoma.

Preferred esters of 3'-azido-3'-deoxythymidine include carboxylic acidesters in which the non-carbonyl moiety of the ester grouping isselected from straight or branched chain alkyl, alkoxyalkyl (e.g.methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g.phenoxymethyl), aryl (e.g. phenyl optionally substituted by halogen,C₁₋₄ alkyl or C₁₋₄ alkoxy); sulphonate esters such as alkyl- oraralkylsulphonyl (e.g. methanesulphonyl); and mono-, di- ortri-phosphate esters. With regard to the above-described esters, unlessotherwise specified, any alkyl moieties present in such estersadvantageously contain 1 to 18 carbon atoms, particularly 1 to 4 carbonatoms. Any aryl moiety present in such esters advantageously comprises aphenyl group. Any reference to any of the above compounds also includesa reference to a pharmaceutically acceptable salt thereof.

Examples of pharmaceutically acceptable salts of3'-azido-3'-deoxythymidine and its pharmaceutically acceptablederivatives include base salts, e.g. derived from an appropriate base,such as alkali metal (e.g. sodium), alkaline earth metal (e.g.magnesium) salts, ammonium and NX+₄ (wherein X is C₁₋₄ alkyl).

3'-azido-3'-deoxythymidine, or a pharmaceutically acceptable derivativethereof (hereafter referred to as the active ingredient), may beadministered to humans in accordance with the invention by any suitableroute including oral, rectal, nasal, topical (including buccal andsublingual), vaginal and parenteral (including subcutaneous,intramuscular, intravenous, intradermal, intramammary, intralesional,intrapleural, intraperitoneal, intrathecal and intra-arterial, and intolymphatic vessels or nodes and to bone or bone marrow).

In general a suitable dose will be in the range of 3.0 to 120 mg perkilogram body weight of the patient per day, preferably in the range of6 to 90 mg per kilogram body weight per day and most preferably in therange 15 to 60 mg per kilogram body weight per day. The desired dose ispreferably presented as two, three, four, five, six or more sub-dosesadministered at appropriate intervals throughout the day. Thesesub-doses may be administered in unit dosage forms, for example,containing 10 to 1500 mg, preferably 20 to 1000 mg, and most preferably50 to 700 mg of active ingredient per unit dosage form. Convenient unitdosage forms contain 250 mg or 500 mg of active ingredient. The dosageused will be at the discretion of the physician and will vary accordingto the route of administration, the nature and severity of thecondition, the immune state and age of the patient, the nature of theactive ingredient, and whether it is used for prophylaxis or treatment.

Experiments with 3'-azido-3'-deoxythymidine suggest that a dose shouldbe administered to achieve peak plasma concentrations of the activecompound of from about 1 to about 75 um, preferably about 2 to 50 um,most preferably about 3 to about 30 um. This may be achieved, forexample, by the intravenous injection of a 0.1 to 5% solution of theactive ingredient, optionally in saline, or orally administered as abolus containing about 1 to about 100 mg/kg of the active ingredient.Desirable blood levels may be maintained by a continous infusion toprovide about 0.01 to about 5.0 mg/kg/hour or by intermittent infusionscontaining about 0.4 to about 15 mg/kg of the active ingredient.

The active ingredients disclosed herein, including3'-azido-3'-deoxythymidine, may also be used in the identification,treatment, prophylaxis, inhibition, alleviation or suppression of othersolid tumours, including adenocarcinomas and carcinomas of humansassociated with retroviral infections.

While it is possible for the active ingredient to be administered alone,it is preferable to present it as a pharmaceutical formulation. Suchformulations comprise at least one active ingredient, as above-defined,together with one or more acceptable carriers thereof and optionallyother therapeutical agents. Each carrier must be "acceptable" in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Formulations include those suitablefor oral, rectal, nasal, topical (including buccal and sublingual),vaginal or parenteral administration. The formulations may convenientlybe presented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. Such methods include the step of bringinginto association the active ingredient with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then if necessary shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. Oral formulations may furtheras sweeteners, flavouring agents and thickners.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatine, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g. sodium starchglycollate, cross-linked povidone, cross-linked sodium carboxymethylcellulose), surface-active or dispersing agent. Moulded tablets may bemade by moulding in a suitable machine a mixture of the powderedcompound moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored.

The above formulations may be formulated so as to provide slow orcontrolled release of the active ingredient therein using, for example,hydroxypropylmethylcellulose in varying proportions to provide thedesired release profile.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose sealed containers, for example, ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above receited, or an appropriatefraction thereof, of an active ingredient.

Specific examples of the above pharmaceutical formulations are describedin European Patent Specification No. 196185 which is herein incorporatedby reference.

In addition to 3'-azido-3'-deoxythymidine and its pharmaceuticallyacceptable derivatives, the present invention also relates to otherknown retroviral reverse transcriptase inhibitors including:phosphonoformic acid, 2',3'-dideoxynucleosides such as2',3'-dideoxycytidine, 2',3'-dideoxyadenosine, 2',3'-dideoxyinosine, and2',3'-dideoxyguanosine.

In accordance with the present invention any such additional reversetranscriptase inhibitor may be employed in place of or in addition to3'-azido-3'-deoxythymidine in the above-described embodiments of theinvention.

The scope and significance of the present invention is more fullyindicated in greater detail, but by way of example only, in thefollowing experimental details:

EXAMPLE 1 Methods Patients and Controls

Monocytes from 32 women with early breast cancer and from 27 healthyage-matched female volunteers with no evidence or family history ofbreast disease were studied. Neither patients nor controls werereceiving any form of medication at the time of study. Diagnosis ofbreast cancer was based on needle biopsy pre-operatively and confirmedon histological examination of the excised tumour.

Monocyte Separation

Peripheral blood monocytes from patients and controls were collected andpurified over Ficoll-Hypaque and a discontinous Percoll densitygradient. See, for example, Al-Sumidiae et al., "Characterisation of theunder agarose method for quantifying migration of highly purified humanmonocytes", Journal of Immunological Methods, 1984, 75 129-40.

Preparation of Cell-free Culture Medium (CFCM)

1 million monocytes from patients and controls were suspended in Eagle'smedium supplemented with 10% fetal calf serum and 15 μmol/15'-azacytidine. After 6 days' incubation at 37° C. in 5% carbon dioxide,in air, in a humidified incubator, the supernatant was filtered througha 220 nm filter. The filtrates were centrifuged at 100000 g for o1 hourat 4° C. The pellets were suspended in 1 ml of TNE buffer pH 8.3 (10mmol/l "tris" - HCl, 150 mmol/l NaCl, 2 mmol/l edetic acid) for reversetranscriptase assay or in 2% phosphotungstic acid for electronmicroscopy.

Reverse Transcriptase Assay

Reverse transcriptase activity was detected by the incorporation ofradiolabelled deoxycytidine triphosphate (dCTP) into DNA in the presenceof a synthetic RNA template. The standard method and template(polyguanylic acid) described by Green et al., "RNA directed DNApolymerase", Prog. Nucl. Acid Res. Mol. Biol., 1974, 14, 202-334 wasused.

To ensure release of RT activity from presumtive retrovirus particles,the high-speed pellet was suspended in `Nonidet P 40` (0.2%, v/v) and 50μmol/l dithiothreitol (DTT) and incubated at 20° C. for 15 min. Theassay reaction mix contained, in a final volume of 100 μl, 45 μl ofsample, 5 μmol "tris" - hydrochloric acid pH 8.3, 5 μmol potassiumchloride, 2.5 μmol DTT, 0.6 μmol magnesium chloride, 0.16 μmol each ofdeoxyadenosine triphosphate, deoxythymidine triphosphate, anddeoxyguanosine triphosphate, 0.05 μmol dCTP, 5 μCi (alpha-³² P) dCTP(3000 ci/mmol), 0.5 μg oligodeoxycytidylic acid (oligo d(pC) 8), and 0.5μg polyguanylic acid. The reaction was incubated at 37° C. for 2 hours.Background incorporation was determined any substituting 45 μl of TNE(preincubated with Nonidet P40 DTT) for the sample in the reactionmixture. The reaction was stopped by the addition of 0.4 ml of 10% (w/v)trichloracetic acid (TCA) and 25 μg of calf thymus DNA as carrier. TheDNA was precipitated overnight at -20° C. The precipitated radioactivitywas collected by filtration onto a GF/C glass-fibre filter and washedwith 50 ml of 5% (w/v) TCA. The radioactivity on the filter was measuredin a scintillation counter.

Sucrose Gradient

A discontinuous density gradient of 20, 30, 40 and 60% sucrose in TNEwas prepared and allowed to stand at 20° C. for 2 hours. This produced arange of densities (1.1-1.28 g/ml) which span the known buoyantdensities (1.16-1.18 g/ml) of retroviruses. A high-speed pellet wasprepared from CFCM of incubated monocytes from patients with breastcancer as described above. The pellet was either distrupted by theaddition of the non-ionic detergents Nonidet P40 and DTT or resuspendedwith NE buffer and incubated at 20° C. for 15 minutes before beinglayered onto the gradient which was then certrifuged at 120,000 g for 16hours at 4° C. (Beckman SW 65 rotor). Fractions (250 μl) were collectedby piercing the bottom of the tube and assayed for RT activity. Thedensity of the fractions was determined with a refractometer.

Electron Microscopy

Tumour tissue and monocytes from patients with breast cancer andmonocytes from control subjects were fixed with cacodylate bufferedglutaraldehyde (2.5% v/v), embedded in araldite, and thin-sectioned.Sections were stained with lead citrate and uranyl acetate (2% v/v).These and the resuspended pellets from CFCM were examined with aPhillips 301 electron microscope.

a. MCF7 cell line

The supernatant from MCF7 cells was assayed for RT activity.

b. U937 cell line

U937 is a non-adherent human monocyte derived continous cell linegrowing in standard RPMI medium. Resuspended pellets derived frommonocytes of patients with breast cancer were added to flasks containingU937 and incubated for 48 hrs. The medium was then replaced and cultureof the U937 cells continued. Two further changes of medium took place atweekly intervals and after a further week of culture the medium wasremoved, filtered through a 200 nm filter and centrifuged at 100,000 gat 4,C for o1 hr. RT assay was then performed on the pellet. Culture ofthe U937 cells was continued with the addition of 15 μmol/l5'azacytidine to the medium and after a further week the medium wasremoved and assayed for RT activity. RT activity was also estimated onU937 cells which had not been treated with monocyte derived material.

c. Cocultivation experiments

Monocytes which were known to be RT positive were added to cultureflasks containing U937 cells. The monocytes adhered to the flask andafter 48 hrs incubation the U937 cells were decanted. The U937 cellswere then treated as before with weekly changes of medium and after thethird change of medium the medium was assayed for RT activity.

Infected U937 cells were then cocultivated with the MRC-5 cell line andafter incubation decanted. Culture of the MRC-5 line was then continuedas usual.

d. Pleural effusions and ascites

Pleural effusions were obtained from 5 patients with secondary breastcancer and ascitic fluid from 1 patient with secondary breast cancer.The cells were separated by centrifugation and cultured in MEM with theaddition of 15 μmol 5'azacytidine. After 6 days incubation thesupernatant was assayed for reverse transcriptase activity. When thecells were established as monolayers, U937 cells were added and culturedfor 48 hrs. The U937 cells were then decanted, cultured and assayed forRT activity as before.

The epithelial origin of the pleural effusion cells was verified bystaining with the epithelial specific monoclonal antibody CAM 5-2.

RESULTS

In the presence of 5'-azacytidine, and taking a cut-off for positivityof 15 pmol of dCTP RT activity was observed in 31 out of 32 patientswith breast cancer (97%). In contrast, RT activity was detected in only3 out of the 27 controls (11%). the mean RT activity of the CFCM frompatients with breast cancer was 732 (SEM 157) pmol of dCTPincorporated/10 monocytes compared with that of control subjects, whichwas 6.5 (SEM 2) pmol of dCTP incorporated/10 monocytes (p<0.0001;Wilcoxon rank sum test, two tailed).

The RT activity detected in the CFCM was found in fractions with buoyantdensities between 1.165 and 1.18 g/ml on a sucrose density gradient.This peak of activity was abolished when the CFCM was treated withNonidet P40 and DTT before separation on the sucrose density gradient.

Monocytes from patients with breast cancer revealed retrovirus-likeparticles near the surface of the cells. These resembled particles seenin HT/H9 cell-line infected with human immunodeficiency virus (HIV), atypical retrovirus used for comparison. Electron microscopy of thebreast cancer cells did not reveal any particles suggestive of viruses,but macrophages within the tumour contained particles similar to thoseobserved in the incubated monocytes from patients with breast cancer andto the HIV in the infected HT/H9 cell line. Negative-stain electronmicroscopy of pellets obtained from CFCM from patients with breastcancer revealed the presence of envelope particles with a fringedsurface resembling murine mammary tumour virus.

MCF7 cell line

RT activity was detected in low titre in the culture of MCF7 which wastested.

U937 cell line

When the supernatant from infected monocytes is added to U937 cells,these cells become producers of reverse transcriptase indicating thatthey have become infected with the retrovirus. These cells are modifiedby this infection and are no longer immortal, dying after 3-4 passages.

Cocultivation of infected monocytes with U937 also results in transferof viral activity to the cell line. This activity is in turn transmittedto MRC-5 which continues to grow without inhibition and appears to be astable producer of virus.

Pleural effusions and ascites

5 pleural effusions and one ascitic fluid have been cultured andassayed. Reverse transcriptase activity has been detected in the culturemedium in all cases. The cells which grow are varied in appearance butstain with CAM 5-2, confirming their epithelial origin. When U937 cellsare cocultivated with these cells the U937 cells become producers ofreverse transcriptase indicating that they have become infected with theretrovirus from the pleural effusion.

EXAMPLE 2 Inhibition of Reverse Transcriptase Activity

Monocytes from a patient with breast cancer were incubated in Eaglesmedium containing 10% foetal calf serum and 1 μM of3'-azido-3'-deoythymidine for 6 days. Cell-free culture medium wasassayed for reverse transcriptase activity in accordance with theprocedures described above and no activity was detected.

    ______________________________________                                                           -AZT  +AZT                                                 ______________________________________                                        Reverse Transcriptase Activity                                                                     1455    222                                              (cpm Incorporated)                                                            ______________________________________                                    

The following Examples are intended for illustration only and are notintended to limit the scope of the invention in any way. The term`active ingredient` as used in the Examples means3'-azido-3'-deoxythymidine.

EXAMPLE 3 Tablet Formulations

The following formulations A to C were prepared by wet granulation ofthe ingredients with a solution of povidone, followed by addition ofmagnesium stearate and compression.

    ______________________________________                                        Formulation A                                                                                        mg/tablet  mg/tablet                                   ______________________________________                                        (a)  Active Ingredient 250        250                                         (b)  Lactose B.P.      210        26                                          (c)  Povidone B.P.     15         9                                           (d)  Sodium Starch Glycollate                                                                        20         12                                          (e)  Magnesium Stearate                                                                              5          3                                                                  500        300                                         ______________________________________                                        Formulation B                                                                                        mg/tablet  mg/tablet                                   ______________________________________                                        (a)  Active Ingredient 250        250                                         (b)  Lactose           150        --                                          (c)  Avicel PH 101     60         26                                          (d)  Povidone B.P.     15         9                                           (e)  Sodium Starch Glycollate                                                                        20         12                                          (e)  Magnesium Stearate                                                                              5          3                                                                  500        300                                         ______________________________________                                        Formulation C                                                                                  mg/tablet                                                    ______________________________________                                        Active Ingredient                                                                              100                                                          Lactose          200                                                          Starch           50                                                           Povidone         5                                                            Magnesium Stearate                                                                             4                                                                             359                                                          ______________________________________                                    

The following formulations, D and E, were prepared by direct compressionof the admixed ingredients. The lactose used in formulation E was of thedirect compression type (Diary Crest--"Zeparox").

    ______________________________________                                                          mg/tablet                                                   ______________________________________                                        Formulation D                                                                 Active Ingredient   250                                                       Pregelatinised Starch NF15                                                                        150                                                                           400                                                       Formulation E                                                                 Active Ingredient   250                                                       Lactose             150                                                       Avicel              100                                                                           500                                                       ______________________________________                                    

Formulation F (Controlled Release Formulation)

The formulation was prepared by wet granulation of the ingredients(below with a solution of povidone followed by the addition of magnesiumstearate and compression.

    ______________________________________                                                            mg/tablet                                                 ______________________________________                                        (a)    Active Ingredient  500                                                 (b)    Hydroxypropylmethylcellulose                                                                     112                                                        (Methocel K4M Premium)                                                 (c)    Lactose B.P.       53                                                  (d)    Povidone B.P.C.    28                                                  (e)    Magnesium Stearate 7                                                                             700                                                 ______________________________________                                    

Drug release took place over a period of about 6-8 hours and wascomplete after 12 hours.

EXAMPLE 4 Capsule Formulations Formulation A

A capsule formulation was prepared by admixing the ingredients ofFormulation D in Example 3 above and filling into a two-part hardgelatin capsule. Formulation B (infra) was prepared in a similar manner.

    ______________________________________                                                           mg/capsule                                                 ______________________________________                                        Formulation B                                                                 (a)     Active Ingredient                                                                              250                                                  (b)     Lactose B.P.     143                                                  (c)     Sodium Starch Glycollate                                                                       25                                                   (d)     Magnesium Stearate                                                                             2                                                                             420                                                  Formulation C                                                                 (a)     Active Ingredient                                                                              250                                                  (b)     Macrogol 4000 BP 350                                                                           600                                                  ______________________________________                                    

Capsules were prepared by melting the macrogol 4000 BP, dispersing theactive ingredient in the melt and filling the melt into a two-part hardgelatin capsule.

    ______________________________________                                        Formulation D                                                                               mg/capsule                                                      ______________________________________                                        Active Ingredient                                                                             250                                                           Lecithin        100                                                           Arachis Oil     100                                                                           450                                                           ______________________________________                                    

Capsules were prepared by dispersing the active ingredient in thelecithin and arachis oil and filling the dispersion into soft, eleasticgelatin capsules.

Formulation E (Controlled Release Capsule)

The following controlled release capsule formulation was prepared byextruding ingredients a, b and c using an extruder, followed byspheronisation of the extrudate and drying. The dried pellets were thencoated with release-controlling membrane (d) and filled into atwo-piece, hard gelatin capsule.

    ______________________________________                                                           mg/capsule                                                 ______________________________________                                        (a)     Active Ingredient                                                                              250                                                  (b)     Microcrystalline Cellulose                                                                     125                                                  (c)     Lactose BP       125                                                  (d)     Ethyl Cellulose  13                                                                            513                                                  ______________________________________                                    

EXAMPLE 5 Injectable Formulation

    ______________________________________                                        Formulation A                                                                 ______________________________________                                        Active Ingredient             0.200 g                                         Hydrochloric acid solution,                                                                     0.1 M q.s. to pH                                                                          4.0 to 7.0                                      Sodium hydroxide solution,                                                                      0.1 M q.s. to pH                                                                          4.0 to 7.0                                      Sterile water     q.s. to     10 ml                                           ______________________________________                                    

The active ingredient was dissolved in most of the water (35°-40° C.)and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid orthe sodium hydroxide as appropriate. The batch was then made up tovolume with the water and filtered through a sterile micropore filterinto a sterile 10 ml amber glass vial (type 1) and sealed with sterileclosures and overseals.

    ______________________________________                                        Formulation B                                                                 ______________________________________                                        Active Ingredient           0.125  g                                          Sterile, pyrogen-free, pH 7 phosphate buffer, q.s. to                                                     25     ml                                         ______________________________________                                    

EXAMPLE 6 Intramuscular Injection

    ______________________________________                                        Active Ingredient       0.20   g                                              Benzyl Alcohol          0.10   g                                              Glycofurol 75           1.45   g                                              Water for Injection q.s to                                                                            3.00   ml                                             ______________________________________                                    

The active ingredient was dissolved in the glycofurol. The benzylalcohol was then added and dissolved, and water added to 3 ml. Themixture was then filtered through a sterile micropore filter and sealedin sterile 3 ml amber glass vials (type 1).

EXAMPLE 7 Ingredients

    ______________________________________                                        Active Ingredient      0.2500  g                                              Sorbitol Solution      1.5000  g                                              Glycerol               2.0000  g                                              Sodium Benzoate        0.0050  g                                              Flavour, Peach 17.42.3169                                                                            0.0125  ml                                             Purified Water q.s. to 5,0000  ml                                             ______________________________________                                    

The active ingredient was dissolved in a mixture of the glycerol andmost of the purified water. An aqueous solution of the sodium benzoatewas then added to the solution, followed by addition of the sorbitolsolution and finally the flavour. The volume was made up with purifiedwater and mixed well.

EXAMPLE 8 Suppository

    ______________________________________                                                               mg/suppository                                         ______________________________________                                        Active Ingredient (63 μm)*                                                                          250                                                  Hard Fat, BP (Witepsol H15 - Dynamit NoBel)                                                            1770                                                                          2020                                                 ______________________________________                                         *The active ingredient was used as a powder wherein at least 90% of the       particles were of 63 μm diameter or less.                             

One-fifth of the Witepsol H15 was melted in a steam-jacketed pan at 45°C. maximum. The active ingredient was sifted through a 200 um sieve andadded to the molten base with mixing, using a silverson fitted with acutting head, until a smooth dispersion was achieved. Maintaining themixture at 45° C., the remaining Witepsol H15 was added to thesuspension and stirred to ensure a homogenous mix. The entire suspensionwas passed through a 250 um stainless steel screen and, with continuousstirring, was allowed to cool to 40° C. At a temperature of 38° C. to40° C. 2.02 g of the mixture was filled into suitable plastic moulds.The suppositories were allowed to cool to room temperature.

EXAMPLE 9 Pessaries

    ______________________________________                                                        mg/pessary                                                    ______________________________________                                        Active Ingredient 63 μm                                                                      250                                                         Anhydrate Dextrose                                                                              380                                                         Potato Starch     363                                                         Magnesium Stearate                                                                               7                                                                            1000                                                        ______________________________________                                    

The above ingredients were mixed directly and pessaries prepared bydirect compression of the resulting mixture.

We claim:
 1. A method for the treatment of breast carcinoma in a humanwhich comprises the administration of a reverse transcriptase inhibitoror a pharmaceutically acceptable derivative thereof to the human in anamount effective to treat said breast carcinoma.
 2. The method accordingto claim 1 wherein said reverse transcriptase inhibitor is selected fromthe group consisting of 3'-azido purine or pyrimidine nucleosides,2',3'-dideoxy purine nucleosides, 3'-fluoro-nucleosides, carbocyclicnucleosides, 2',3'-dideoxy-2',3'-didehydro nucleosides and ribavirin. 3.The method according to claim 1 wherein said reverse transcriptaseinhibitor is 3'-azido-3'-deoxythymidine.
 4. A method of alleviating thesymptoms of human breast carcinoma which comprises the administration ofa reverse transcriptase inhibitor or a pharmaceutically acceptablederivative thereof to the human in an amount effective to alleviate saidsymptoms.
 5. A method of inhibiting the reverse transcriptase activityof retrovirus or retrovirus-like particles in monocytes containing suchparticles which method comprises treating such monocytes with effectiveamounts of 3'-azido-3'-deoxythymidine or a pharmaceutically acceptablederivative thereof.